武装GV1001的重组溶瘤流感病毒构建及抗肿瘤作用研究

Construction and anti-tumor effect validation of recombinant oncolytic influenza virus armed with GV1001

目的拯救嵌合GV1001的重组溶瘤流感病毒株,并通过体内外实验全面评价其选择性杀伤肝癌的效果。方法将编码GV1001肽的碱基序列串联重复3次序列优化设计后插入到A/PuertoRico/8/34(PR8)病毒的NA片段。采用反向遗传学技术,重组质粒pOV-GV1001-NA与pHW191-PB2、pHW192-PB1、pHW193-PA、pHW194-HA、pHW195-NP、pHW197-M、pHW198-NS PR87个质粒共同转染COS 1/MDCK细胞,拯救得到重组溶瘤病毒rOV-GV1001-NA。经过血凝实验、TCID_(50)、PCR、透射电镜、细胞免疫荧光等鉴定,流式细胞术检测rOV-GV1001-NA诱导肝癌细胞死亡的方式,进一步通过肝癌H22细胞小鼠皮下荷瘤模型评价rOV-GV1001-NA体内溶瘤效果。结果重组溶瘤病毒rOV-GV1001-NA血凝效价为2^(9),病毒滴度达5 LogTCID_(50)/ml,并可在SPF鸡胚中稳定传代;电镜观察形态大小符合流感病毒特点;细胞免疫荧光证明GV1001成功插入并表达;细胞凋亡实验表明rOV-GV1001-NA可选择性诱导肝癌细胞凋亡;肝癌小鼠皮下荷瘤模型证实rOV-GV1001-NA可显著抑制肿瘤生长,延长小鼠生存期,且具有剂量依赖性。结论利用反向遗传学技术成功构建了武装GV1001的重组溶瘤流感病毒,体内外实验表明rOV-GV1001-NA能够靶向杀伤肝癌细胞而对正常细胞无明显影响,该结果将为肿瘤疫苗研究提供新思路,有望为肝癌临床治疗开辟新途径。

Oncolytic virus could selectively kill tumors and induce systemic anti-tumor immune response showing broad application prospects in clinic.This study was designed to rescue the chimeric recombinant oncolytic influenza armed with GV1001,and evaluate its effect on selective killing of liver cancer in vitro and in vivo.The sequence encoding GV1001 peptide was optimized in triple tandem repeats and inserted into the NA fragment of A/PuertoRico/8/34(PR8)virus.Using reverse genetics technology,the recombinant plasmid pOV-GV1001-NA and the remaining seven plasmids of the PR8 backbone,containing pHW191-PB2,pHW192-PB1,pHW193-PA,pHW194-HA,pHW195-NP,pHW197-M and pHW198-NS,were co-transfected with COS1/MDCK cells to rescue the recombinant oncolytic virus rOV-GV1001-NA.Furthermore,the recombinant oncolytic virus was identified with hemagglutination,TCID_(50),PCR,transmission electron microscope(TME),cellular immunofluorescence,etc.In vitro,flow cytometry was used to detect the way of rOV-GV1001-NA to induce hepatocellular carcinoma cell death.Furthermore,the oncolytic effect of rOV-GV1001-NA in vivo was evaluated in the subcutaneous H22 tumorbearing mice model.Data showed that hemagglutination titers of recombinant oncolytic virus rOV-GV1001-NA was 27,and the viral titers reached 5 LogTCID_(50)/ml.rOVGV1001-NA could stably passage in SPF chicken embryos.Viral morphology and size observed with TME were similar with the characteristics of influenza virus.Meanwhile,the fluorescent images demonstrated successful insertion and functional expression of GV1001.Apoptosis results showed that rOV-GV1001-NA can selectively induce hepatocellular carcinoma cell apoptosis.In addition,in homologous allogeneic mouse models,we found that rOV-GV1001-NA could significantly inhibit tumor growth and prolong mouse survival in a dose-dependent manner.In conclusion,the recombinant oncolytic influenza virus armed with GV1001 has successfully constructed using reverse genetics technology,while in vivo and in vitro experiments demonstrated that rOV-GV1001-NA could selectively target and kill liver cancer cells without obvious effect on normal cells,which will provide a new idea for cancer vaccines and a novel strategy for cancer treatment.